Abstract The goal of this Trailblazer R21 research proposal is to develop an anticancer chemotherapeutic platform derived entirely from polymerized nucleotides that selectively delivers cytostatic nucleotides to tumor cells. This proposal is motivated by the complexity and low yield of syntheses of current aptamer-targeted nanoparticle drug carriers that are typically carried out by multi-step chemical conjugation of aptamer and drug to a carrier. In contrast, herein we propose a highly efficient in situ enzymatic polymerization strategy that ?grows? a polynucleotide drug segment and a self-assembly segment from an aptamer ?initiator.? This approach exploits our recent discovery that terminal deoxynucleotidyl transferase (TdT), a template independent polymerase, can polymerize 5-fluoro-2'-deoxyuridine (FdUTP), a nucleotide analog of the anticancer drug 5-fluorouracil (5-FU). Building on this finding, we will use TdT catalyzed DNA polymerization (TcEP) to grow a polynucleotide chain that consist of repeats of FdUTP, a cytostatic unnatural nucleotide, from a primer that consists of a DNA or RNA aptamer. The polynucleotide will be terminated with an optional segment that contains multiple copies of hydrophobic, unnatural nucleotides that drive self-assembly of the construct into micelles. Our ?one-pot? enzymatic reaction approach offers a new, dramatically simplified and innovative route for the design and synthesis of an ?all nucleotide? drug delivery platform. The central hypothesis of this proposal is that an aptamer-functionalized poly(FdU) nanoparticle will be a highly potent anticancer agent with improved cancer targeting, drug delivery and efficacy compared to the free drug analog, 5-FU. In Specific Aim 1, we will synthesize poly(FdU) ?a polymer drug? from an aptamer initiator. We then investigate poly(FdU)'s cell uptake and compare its in vitro cytotoxicity with that of its free drug analog ?5-FU. Building upon these studies, in Specific Aim 2, we will use a sequential TcEP reaction to synthesize amphiphilic block co- polynucleotides that contain an aptamer targeting motif, a poly(FdU) drug block, and a block that contains multiple copies of unnatural hydrophobic nucleotides that drive self-assembly into micelles. We will compare the cytotoxicity of these nanoparticles with poly(FdU) and 5-FU, investigate their cellular uptake in tumor cells that overexpress the target receptor for the aptamer, and investigate their in vivo pharmacokinetics (PK), biodistribution, and antitumor efficacy, in a mouse tumor xenograft model. The impact of this research will be the development of a polynucleotide cancer drug delivery platform that has several useful features: (i) easy design and synthesis of aptamer targeted nanoparticles, (ii) tunable nucleotide-drug loading capacity, and (iii) targeting versatility by choice of aptamer that target a tumor type of interest. If successful, this research will show greatly improved therapeutic anti-cancer efficacy of targeted nanoparticles compared to free drug, thus positioning this platform technology as a blueprint for a new architecture in nanomedicine.